Inna Aphasizheva

Isolation of a U‐insertion/deletion editing complex from Leishmania tarentolae mitochondria

A multiprotein, high molecular weight complex active in both U‐insertion and U‐deletion as judged by a pre‐cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem affinity purification (TAP) procedure, using three different TAP‐tagged proteins of the complex. This editing‐ or E‐complex consists of at least three protein‐containing components interacting via RNA: the RNA ligase‐containing L‐complex, a 3′ TUTase (terminal uridylyltransferase) and two RNA‐binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identified by mass spectrometric analysis of the core L‐complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids.

Trypanosome mitochondrial 3' terminal uridylyl transferase (TUTase): the key enzyme in U-insertion/deletion RNA editing.

A 3 terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3 U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.

A tale of two TUTases

The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3′ terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3′ oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purification-tagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3′ end of a single-stranded RNA, thereby confirming that RET2 is a 3′ TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro U-insertion editing and has no effect on the length of the 3′ oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3′ end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.

Guide RNA-Binding Complex from Mitochondria of Trypanosomatids

In the mitochondria of trypanosomatids, the majority of mRNAs undergo massive uracil-insertion/deletion editing. Throughout the processes of pre-mRNA polyadenylation, guide RNA (gRNA) uridylylation and annealing to mRNA, and editing reactions, several multiprotein complexes must engage in transient interactions to produce a template for protein synthesis. Here, we report the identification of a protein complex essential for gRNA stability. The gRNA-binding complex (GRBC) interacts with gRNA processing, editing, and polyadenylation machineries and with the mitochondrial edited mRNA stability (MERS1) factor. RNAi knockdown of the core subunits, GRBC1 and GRBC2, led to the elimination of gRNAs, thus inhibiting mRNA editing. Inhibition of MERS1 expression selectively abrogated edited mRNAs. Homologous proteins unique to the order of Kinetoplastida, GRBC1 and GRBC2, form a stable 200 kDa particle that directly binds gRNAs. Systematic analysis of RNA-mediated and RNA-independent interactions involving the GRBC and MERS1 suggests a unified model for RNA processing in the kinetoplast mitochondria.

3′ adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post‐transcriptionally and requires extensive U‐insertion/deletion mRNA editing. In mitochondrial extracts, 3′ adenylation reportedly influences degradation kinetics of synthetic edited and pre‐edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A‐tails typically found in mitochondrial mRNAs, and decreases the abundance of never‐edited and edited transcripts. Pre‐edited mRNAs are not destabilized by the lack of 3′ adenylation, whereas short A‐tails are required and sufficient to maintain the steady‐state levels of partially edited, fully edited, and never‐edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A‐tail in edited molecules. Upon completion of the editing process, the short A‐tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3′ processing and editing of mitochondrial mRNAs in trypanosomes.

Pentatricopeptide Repeat Proteins Stimulate mRNA Adenylation/Uridylation to Activate Mitochondrial Translation in Trypanosomes

The majority of trypanosomal mitochondrial pre-mRNAs undergo massive uridine insertion/deletion editing, which creates open reading frames. Although the pre-editing addition of short 3 A tails is known to stabilize transcripts during and after the editing, the processing event committing the fully edited mRNAs to translation remained unknown. Here, we show that a heterodimer of pentatricopeptide repeat-containing (PPR) proteins, termed kinetoplast polyadenylation/uridylation factors (KPAFs) 1 and 2, induces the postediting addition of A/U heteropolymers by KPAP1 poly(A) polymerase and RET1 terminal uridyltransferase. Edited transcripts bearing 200- to 300-nucleotide-long A/U tails, but not short A tails, were enriched in translating ribosomal complexes and affinity-purified ribosomal particles. KPAF1 repression led to a selective loss of A/U-tailed mRNAs and concomitant inhibition of protein synthesis. These results establish A/U extensions as the defining cis-elements of translation-competent mRNAs. Furthermore, we demonstrate that A/U-tailed mRNA preferentially interacts with the small ribosomal subunit, whereas edited substrates and complexes bind to the large subunit.

Uridine insertion/deletion editing in trypanosomes: a playground for RNA-guided information transfer.

RNA editing is a collective term referring to enzymatic processes that change RNA sequence apart from splicing, 5′ capping or 3′ extension. In this article, we focus on uridine insertion/deletion mRNA editing found exclusively in mitochondria of kinetoplastid protists. This type of editing corrects frameshifts, introduces start and stops codons, and often adds much of the coding sequence to create an open reading frame. The mitochondrial genome of trypanosomatids, the most extensively studied clade within the order Kinetoplastida, is composed of ∼50 maxicircles with limited coding capacity and thousands of minicircles. To produce functional mRNAs, a multitude of nuclear‐encoded factors mediate interactions of maxicircle‐encoded pre‐mRNAs with a vast repertoire of minicircle‐encoded guide RNAs. Editing reactions of mRNA cleavage, U‐insertions or U‐deletions, and ligation are catalyzed by the RNA editing core complex (RECC, the 20S editosome) while each step of this enzymatic cascade is directed by guide RNAs. These 50–60 nucleotide (nt) molecules are 3′ uridylated by RET1 TUTase and stabilized via association with the gRNA binding complex (GRBC). Remarkably, the information transfer between maxicircle and minicircle transcriptomes does not rely on template‐dependent polymerization of nucleic acids. Instead, intrinsic substrate specificities of key enzymes are largely responsible for the fidelity of editing. Conversely, the efficiency of editing is enhanced by assembling enzymes and RNA binding proteins into stable multiprotein complexes. WIREs RNA 2011 2 669–685 DOI: 10.1002/wrna.82

RET1-Catalyzed Uridylylation Shapes the Mitochondrial Transcriptome in Trypanosoma brucei

ABSTRACT RNA uridylylation is critical for the expression of the mitochondrial genome in trypanosomes. Short U tails are added to guide RNAs and rRNAs, while long A/U heteropolymers mark 3′ ends of most mRNAs. Three divergent mitochondrial terminal uridylyl transferases (TUTases) are known: RET1 catalyzes guide RNA (gRNA) uridylylation, RET2 executes U insertion mRNA editing, and MEAT1 associates with the editosome-like complex. However, the activities responsible for 3′ uridylylation of rRNAs and mRNAs, and the roles of these modifications, are unclear. To dissect the functions of mitochondrial TUTases, we investigated the effects of their repression and overexpression on abundance, processing, 3′-end status, and in vivo stability of major mitochondrially encoded RNA classes. We show that RET1 adds U tails to gRNAs, rRNAs, and select mRNAs and contributes Us into A/U heteropolymers. Furthermore, RET1s TUTase activity is required for the nucleolytic processing of gRNA, rRNA, and mRNA precursors. The U tails presence does not affect the stability of gRNAs and rRNAs, while transcript-specific uridylylation triggers 3′ to 5′ mRNA decay. We propose that the minicircle-encoded antisense transcripts, which are stabilized by RET1-catalyzed uridylylation, may direct a nucleolytic cleavage of multicistronic precursors.

Mitochondrial RNA processing in trypanosomes

The mitochondrial genome of trypanosomes is composed of ∼50 maxicircles and thousands of minicircles. Maxi-(∼25xa0kb) and mini-(∼1xa0kb)circles are catenated and packed into a dense structure called a kinetoplast. Both types of circular DNA are transcribed by a phage-like RNA polymerase: maxicircles yield multicistronic rRNA and mRNA precursors, while guide RNA (gRNA) precursors are produced from minicircles. To function in mitochondrial translation, pre-mRNAs must undergo a nucleolytic processing and 3 modifications, and often uridine insertion/deletion editing. gRNAs, which represent short (50-60xa0nt) RNAs directing editing reactions, are produced by 3 nucleolytic processing of a much longer precursor followed by 3 uridylation. Ribosomal RNAs are excised from precursors and their 3 ends are also trimmed and uridylated. All tRNAs are imported from the cytoplasm and some are further modified and edited in the mitochondrial matrix. Historically, the fascinating phenomenon of RNA editing has been extensively studied as an isolated pathway in which nuclear-encoded proteins mediate interactions of maxi- and minicircle transcripts to create open reading frames. However, recent studies unraveled a highly integrated network of mitochondrial genome expression including critical pre- and post-editing 3 mRNA processing, and gRNA and rRNA maturation steps. Here we focus on RNA 3 adenylation and uridylation as processes essential for biogenesis, stability and functioning of mitochondrial RNAs.

Mitochondrial RNA editing in trypanosomes: small RNAs in control

Mitochondrial mRNA editing in trypanosomes is a posttranscriptional processing pathway thereby uridine residues (Us) are inserted into, or deleted from, messenger RNA precursors. By correcting frameshifts, introducing start and stop codons, and often adding most of the coding sequence, editing restores open reading frames for mitochondrially-encoded mRNAs. There can be hundreds of editing events in a single pre-mRNA, typically spaced by few nucleotides, with U-insertions outnumbering U-deletions by approximately 10-fold. The mitochondrial genome is composed of ∼50 maxicircles and thousands of minicircles. Catenated maxi- and minicircles are packed into a dense structure called the kinetoplast; maxicircles yield rRNA and mRNA precursors while guide RNAs (gRNAs) are produced predominantly from minicircles, although varying numbers of maxicircle-encoded gRNAs have been identified in kinetoplastids species. Guide RNAs specify positions and the numbers of inserted or deleted Us by hybridizing to pre-mRNA and forming series of mismatches. These 50-60 nucleotide (nt) molecules are 3 uridylated by RET1 TUTase and stabilized via association with the gRNA binding complex (GRBC). Editing reactions of mRNA cleavage, U-insertion or deletion, and ligation are catalyzed by the RNA editing core complex (RECC). To function in mitochondrial translation, pre-mRNAs must further undergo post-editing 3 modification by polyadenylation/uridylation. Recent studies revealed a highly compound nature of mRNA editing and polyadenylation complexes and their interactions with the translational machinery. Here we focus on mechanisms of RNA editing and its functional coupling with pre- and post-editing 3 mRNA modification and gRNA maturation pathways.